The human DNA repair enzyme o-6 methylguanine DNA methyltransferaase (MGMT) is a major determinant in the sensitivity of tumors to therapeutic chloroethylnitrosoureas (CENU). The long term objective of this proposal is to enhance the therapeutic efficacy of by understanding ) and manipulating the regulation of expression of gene. Preliminary studies suggest that regulation of expression of the MGMT gene is associated with DNA cytosine methylation, but in a paradoxically direct fashion, i.e. hypermethylation of regions gene is associated with gene expression rather than inactivation. The proposed research will therefore evaluate the role of DNA methylation in MGMT expression, and will test the hypothesis that demethylation of regions of the MGMT gene results in decreased MGMT expression. This will be accomplished through the following 5 specific 1) To clone regions of the MGMT gene likely to be involved in methylation-related regulation-of MGMT expression; 2) To determine if methylation status of these MGMT gene regions correlates with MGMT expression; 3) To determine if de-methylating expression- related regions of the MGMT gene alters cellular MGMT gene expression and BCNU sensitivity; 4) To determine if methylation-related MGMT regulatory regions identified in human tumor cell lines are relevant in human tumors; 5) To assess the role of DNA binding proteins as mediators of relationship between MGMT gene methylation and MGMT expression. studies are likely to contribute to the understanding of both MGMT regulation, and the role of DNA methylation in gene expression. in turn may aid in the development of therapeutic strategies targeting DNA methylation and methylation-related DNA binding proteins as a means of reducing MGMT expression and enhancing CENU efficacy.